An important component of the AIDS pandemic is pediatric infection. Each year, hundreds of thousands of children become infected with HIV. At least 90% of pediatric infections are a result of mother-to-child transmission (MTCT). Increased rates of miscarriage also are associated with HIV infection. The FIV-infected cat is an excellent small-animal model of MTCT. Very high rates of placental and fetal infection occur in cats experimentally infected with FIV-B-2542, and reproductive failure is frequent at early and late-term pregnancy. Preliminary analyses of placental immune parameters indicate that inflammation accompanies reproductive failure. Placental regulatory T cells (Tregs) are centrally-important in the maintenance of pregnancy because their immunosuppressive function allows maternal immune tolerance to the semi-allogeneic fetus. Depletion of Tregs is associated with reproductive failure. FIV preferentially replicates in this phenotype of T cells in the infected cat, thereby causing Treg depletion during chronic infection and consequent hyperimmune activation. This information supports the following hypotheses: 1) Altered population dynamics and function of placental Tregs occurs as a result of FIV infection of these cells. 2) This immunopathological effect of infection results in the failure to suppress inflammation at the maternal-fetal interface and contributes to viral MTCT and/or compromised pregnancy. Two aims will be addressed in this project: 1) Determine Treg population dynamics and activation at early and late pregnancy in infected and control cats;2) Examine the placental microenvironment proximate to Tregs to determine whether expression of immunomodulators is affected by the Treg activation state. Frozen or formalin-fixed placental tissues from early and late gestation will be used. Treg cells will be identified in placental sections by immunofluorescent labeling with antibodies to CD4, CD25, and FoxP3. Confocal microscopy will be used to quantify these cells. Immunohistochemistry will be done to label Tregs for laser capture microdissection (LCM). LCM will be performed to microdissect Tregs, along with tissue from the proximate microenvironment. RNA will be purified from microdissected tissues. Target gene and b- actin expression will be quantified using real time reverse-transcriptase (RT)-PCR. Treg activation markers (FoxP3, CTLA4, TGF-b, B7, CD25), receptors (CD134 and CXCR4) and cytokines (IL-2 and IL-10) will be quantified from the Treg RNA. FIV RNA load in these cells also will be quantified. The immunomodulators that will be evaluated in the adjacent microenvironment will include indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme produced by antigen presenting cells (APCs) and trophoblasts, cytokines INF-c, TNF-", IL-1b, IL-2, IL-6, and IL-12, produced by APCs, cytotoxic T cells, and NK cells. This study should reveal the role of placental Treg function in MTCT of FIV and reproductive failure in the feline model, thus broadening our understanding of fetal infection and reproductive outcome in HIV-infected women. PUBLIC HEALTH RELEVANCE: Pediatric HIV infections are largely a result of maternal-fetal transmission, and miscarriages occur at an elevated rate in HIV-infected women. Regulatory T cells (immunosuppressive T cells) in the placenta are known to support successful pregnancy by allowing maternal tolerance of the developing fetus, yet HIV can perturb regulatory T cell function. The purpose of this project is to evaluate the effect of HIV infection on the function of regulatory T cells, using the feline immunodeficiency virus (FIV)-infected cat to model maternal-fetal infection and compromised pregnancy.